1. Technical Field
The present invention is related to a method and device for quantitative determination of fluorescent reaction product endpoint in immunofluorescence using microfluorophotometry. More particularly, the present invention is related to standardization of various parameters, calibration of components or devices and determination of reaction conditions for a reliable, stable and reproducible quantitation of fluorescent reaction product in immunofluorescent microscopy or microfluorophotometry.
2. Prior Art
Current method for immunofluorescent (IF) test involves a subjective evaluation of the end point (titer) which is dependent, inter alia, upon the observer's expertise, experience and judgment. This subjectivity is further complicated by the rapid fading of the fluorescent reaction product (FRP) under the test conditions routinely employed. Thus, as the art is presently known, the outcome of an IF test becomes a function of time and judgment. In fact, there being no better or objective method for IF assay, researchers have generally conceded that rapid fading of fluorophores would have to be tolerated if IF is opted as the procedure of choice. Nairn et al., Clin. Exp. Immunol. 4, 697-705 (1969); Johnson et al., J. Immunol. Meth. 55, 231-242 (1982); Schauenstein et al., J. Immunol. Meth. 8, 9-16 (1975); Wick et al., Ann. New York Acad. Sci. 254, 172-174 (1975); McKay et al., Immunology 43, 591-602 (1981).
Various techniques have been used to protect the sample from fading. These are summarized in Table 1.
TABLE 1 ______________________________________ METHODS FOR THE REDUCTION OF FADING Technique Investigator ______________________________________ (1) Localization under phase Ploem, Golden, Fukuda, Geyer contrast (2) Fast, epi-shutter Ploem, Golden, Geyer, Kaufmann excitation Nairn (3) Chemical Agents Gill, Johnson, Sedat, Giloh, Kaplan, Picciolo (4) Pre- or Post-illumination Fukuda, Fujita (5) Variable iris diaphragm Golden, Ploem on objective (6) Neutral density filters Nairn, Ploem (7) Light sources Goldman, Haaijman, Johnson (8) Excitor/barrier filters Goldman, Haaijman, Nairn, McKay (9) Field diaphragms Golden, Ploem, Haaijman ______________________________________
Of these, a more practical and feasible technique appears to be the use of chemical additives in the mounting medium to protect the fluorophore from the effects of the excitation light.
Protection from fading would make exposure of the specimen to the exciting light less critical. This would allow ease in the localization of the fluorescent specimens and permit more accurate discrimination between weakly positive and negative results, which is difficult if the sample is rapidly fading.
Certain tests, such as determination of the type of Herpes present, require finding any positive cells that may be present on the entire slide. This searching procedure may take several minutes and must be done during excitation to recognize the presence of the positives. If fading is rapid, positives may be missed. Protection from fading in these cases is critical.
Reducing the fading would also significantly improve quantitation of the FRP on the IF microscopy slides. Retarding fading would permit longer scan times on slides without concomitant decrease in fluorescence intensity. This would permit the use of automated or semi-automated instrumentation which could scan a slide and determine the endpoint quantitatively.
Rapidly fading specimens account for many false negatives in the clinical laboratory. In some cases, by the time the technician has set up the slide on the microscope, the weakly positive cell has faded to a negative cell. In the case of antinuclear antibody (ANA) positive cells, the technician cannot properly identify the staining pattern if the specimen is rapidly fading. Stabilization of the fluorescence emission is, therefore, necessary for objective and quantitative determination of antibody level.
A factor which must also be considered in the evaluation of the IF assay, is the variability due to the instrumentation.
Earlier methods used microcapillaries filled with the fluorophore of interest. In this method the microcapillary diameter is measured microinterferometrically, allowing exact calibration of the microscope fluorometer and correlation of the measured fluorescence intensities with the mass of the excited fluorochrome. Serntz, et al., Fluorescence Techniques in Cell Biology Springer-Verlag, N.Y., pp 41-49 (1973). Certain applications may not require an exact calibration, but an easier-to-perform secondary standardization may be satisfactory.
Recent work by Jensen et al., J. Immuno. Meth. 42: 343-353 (1981) used a particle of europium salt phosphor as a daily standard.
The Applicants have now improved the immuno-fluorescent methodologies by various means amongst which are: (a) removing the subjective evaluation of endpoint determination by providing a quantitative measure; (b) providing a reagent-reactivity-monitor which includes performance testing associated with a numerical value, (c) providing a calibration standard for inter-laboratory brightness comparison; and (d) achieving a quantitative measure by a fading retardant means.